DESCRIPTION (APPLICANT'S ABSTRACT): The secretory pathway is central to the interaction of cells with their environment as it is the route by which proteins and peptides are released into the surrounding melieu, receptors, ion channels and ion pumps are presented on the plasma membrane, and ligands and receptors are internalized. Elucidation of the mechanisms that regulate this pathway is relevant for a variety of diseases that are caused by impaired transport of a substance that is either essential as insulin in diabetes and CFTR in cystic fibrosis, or detrimental as beta-amyloid in Alzheimer disease. In the secretory pathway proteins are transported between intracellular compartments via membranous vesicles. Small GTPases play a key role in the regulation of this vesicular trafficking. The mechanisms and machinery that underlie vesicle formation, targeting and fusion are highly conserved between yeast and man. We will use yeast as a model system to address these complicated issues, since this organism allows the use of powerful genetic approaches in addition to molecular and cellular ones. Our long term goal is to understand how Ypt/Rab GTPases regulate and coordinate the discrete steps of the secretory pathway. The proposed research will focus on proteins that function as regulators or effectors for Ypt GTPases. They will be used to address three important questions. 1) What is the role that regulators of Ypt nucleotide cycling play in Ypt-mediated protein transport? Cycling between the GTP- and GDP-bound forms, facilitated by accessory factors, is considered crucial for Ypt/Rab function. However, little is known regarding how these factors locate their Ypt/Rab targets, facilitate nucleotide exchange or hydrolysis, and affect Ypt GTPase function. To resolve these issues, the localization of regulators, their function and their interaction with Ypt GTPases will be examined both in vivo and in vitro. 2) Do Ypt proteins function in GTPase cascades? This existing hypothesis suggests that Ypt proteins affect nucleotide cycling of other GTPases to coordinate discrete steps of the secretory pathway. The existence and the nature of GTPase cascades will be first investigated in vitro, and later their role will be tested in vivo. 3) What role do effectors of Ypt GTPases play in protein transport? Ypt/Rab proteins, like other GTPases, are believed to function through the recruitment of down-stream effectors, which transmit signals from the GTP-bound GTPase to the membrane docking and/or fusion apparatus. To determine the mechanism by which Ypt effectors function, we will study the effect of mutations in Ypt effector genes on vesicle budding, targeting, or fusion, and the nature of their interaction with Ypt GTPases.